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(The FASEB Journal. 2000;14:2559-2568.)
© 2000 FASEB

Functional characterization of MT3-MMP in transfected MDCK cells: progelatinase A activation and tubulogenesis in 3-D collagen lattice

TIEBANG KANG, JUN YI, WANGRONG YANG, XING WANG, AIXIANG JIANG and DUANQING PEI1

Department of Pharmacology, University of Minnesota, Minneapolis, Minnesota 55455, USA

1Correspondence: Department of Pharmacology, 6–120 Jackson Hall, 321 Church St. S.E., University of Minnesota, Minneapolis, MN 55455, USA. E-mail: peixx003{at}tc.umn.edu


   ABSTRACT
TOP
 ABSTRACT
INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MT3-MMP, a membrane-anchored matrix metalloproteinase, has been proposed to participate in the remodeling of extracellular matrix either by direct proteolysis or via activating other enzymes such as progelatinase A. To test this hypothesis, we analyzed the effect of exogenously transfected MT3-MMP in a tissue remodeling system: growth and tubulogenesis of Madin-Darby canine kidney (MDCK) cells in collagen gels. Although the parental cells require MMP activities for both growth and tubulogenesis, over-expression of wild-type MT3-MMP, but not its catalytically inactive mutant, leads to further enhancement of both processes, independent of its downstream substrate, progelatinase A. Mechanistically, MT3-MMP accomplishes such an effect by displaying on cell surfaces as active species, ready to activate progelatinase A or degrade ECM molecules. These data strongly suggest that MT3-MMP possesses the potential to directly enhance the growth and invasiveness of cells in vivo, two critical processes for development and carcinogenesis.—Kang, T., Yi, J., Yang, W., Wang, X., Jiang, A., and Pei D. Functional characterization of MT3-MMP in transfected MDCK cells: progelatinase A activation and tubulogenesis in 3-D collagen lattice.


Key Words: ECM • zymogen • membrane-bound MMP • 3-D collagen • tubulogenesis


   INTRODUCTION
TOP
 ABSTRACT
 INTRODUCTION
MATERIALS AND METHODS
 RESULTS
 DISCUSSION
 REFERENCES
 
MATRIX METALLOPROTEINASES (MMP) are a family of well-conserved, zinc-dependent and matrix-degrading endopeptidases implicated in the destruction of extracellular matrix (ECM) during various physiological and pathological processes from embryo development to carcinogenesis (1 2 3) . There are two main categories of MMPs based on cellular localizations: secreted and membrane bound. Whereas the secreted MMPs form a larger group, the newly identified membrane-bound MMPs have attracted considerably more attention, largely because of the discovery of MT1-MMP as the cell-surface activator of progelatinase A and subsequently dubbed as a master switch for extracellular proteolysis (4 5 6) . Currently, there are six members within the membrane-bound subfamily (4 , 7 8 9 10 11 12 13) . Based on sequence alignment, these six MT-MMPs could be further classified into two branches: MT1, 2, 3, 5-MMPs, and MT4, 6-MMPs, respectively (11) . In addition, these two subgroups may differ in membrane-anchoring mechanisms. Itoh and colleagues reported that MT4-MMP may be anchored on the membrane via a novel GPI-linkage (10 , 14) , rather than a traditional transmembrane domain found in MT1, 2, 3, 5-MMPs (4 , 7 , 8 , 12 , 13) . Likewise, the latest addition, MT6-MMP, may be GPI anchored as well (11) . With the expansion of the MT-MMP subgroup, it is apparent that they play a much broader role than simply mediating the activation of progelatinase A as originally proposed (4 , 15) . Indeed, MT1-MMP knockout mice exhibit a phenotype vastly different from those mice deficient in MMP2 (16 , 17) , supporting the idea that MT-MMPs could mediate unique biological processes by expressing intrinsic matrix-degrading activities (18 , 19) . So far, the mechanisms by which MT-MMPs mediate ECM remodeling and determine cellular phenotype have not been fully explored, in a large part because of the lack of suitable experimental systems.

MT3-MMP was originally identified from an oral melanoma (7) , but also detected in brain, lung, placenta, smooth muscle cells, and malignant tumor tissues including renal carcinoma (7 , 20 21 22 23) . Biochemically, MT3-MMP has been shown to be a progelatinase A activator and effective proteinase in degrading various ECM components including native collagens (7 , 24 , 25) . However, the biological consequence of MT3-MMP expression has not been explored so far. Given the recent study by Kitagawa and colleagues that implicated the involvement of MT3-MMP in the invasiveness of renal carcinoma (23) , we rationalize that up-regulated MT3-MMP confers growth and invasiveness to renal tumor cells. To test this possibility, we take advantage of a renal morphogenesis model (26) , which was used by Sato and colleagues to analyze MT1-MMP (27) . Although the depletion of MT1-MMP blocked MDCK cells’ ability to form tubules in collagen gels, overexpression of MT1-MMP did not enhance HGF-induced tubulogenesis, but altered the morphology of the cysts in collagen gels (27) . In contrast, we report here that overexpression of MT3-MMP enhances not only the growth but also tubulogenesis of MDCK cells in collagen gels independent of gelatinase A, a phenotype relevant to renal development and carcinogenesis.


   MATERIALS AND METHODS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
RESULTS
 DISCUSSION
 REFERENCES
 
Cell culture and reagents
MDCK and its derivatives are maintained as described (28) . Cell culture media were purchased from Life Technologies (Rockville, Md.). Restriction enzymes and DNA cloning reagents were from Promega (Madision, Wis.). Rabbit anti-MT3-MMP antisera were raised against a GST-MT3-MMP fusion protein as described (29) . M2 antibody and other immunological reagents were from Sigma (St. Louis, Mo.).

MT-MMP cDNA constructs
For MT3-MMP, two primers, 1256E AGT ATG ATC TTA CTC ACA TTC AGC ACT GGA and B6428 GTC ACT TGT CAT CGT CGT CCT TGT AGT CCA CCC ACT CTT GCA TAG AGC were synthesized according to the published sequence of MT3-MMP (7) and paired to amplify the full-length human MT3-MMP cDNA by RT-PCR from HT1080 cells and cloned into expression vector pCR3.1uni as described (18 , 29) . The resulting clones were verified by DNA sequencing. One error-free clone, designated pCR3.1MT3-MMP, was used throughout this study. Primer B6428 encodes the last seven residues of MT3-MMP followed by sequences for the FLAG tag for convenience of detection using M2 anti-FLAG monoclonal antibody as described (12) . The MT3-MMPEA mutant was created by sequential PCR using two primers at the catalytic motif: GCA GTC CAT GCA CTG GGA CAT, ATG TCC CAG TGC ATG GAC TGC, and cloned into pCR3.1 expression vector as described previously (30) . MT1-MMP and MT5-MMP constructs were described previously (12 , 18) .

DNA transfection and generation of MT3-MMP stable lines
pCR3.1MT3-MMP and pCR3.1MT3-MMPEA were transfected into MDCK cells by LipofectAMINE (Life) and stable clones were selected in the presence of G418 (28) . The stable clones were verified by Western analysis of the cell lysates with M2 antibody and zymographic analysis for their ability to activate progelatinase A present in the serum-containing media. Positive cells were further analyzed by Northern blotting using an MT3-MMP cDNA probe.

Western blotting, immunoprecipitation, and gelatin zymography
The basic protocols for these procedures were carried out as described previously (12 , 29) . For zymography, MDCK and the MT3-MMP stable lines were washed three times with PBS and allowed to incubate in the presence of DMEM supplemented with progelatinase A from either a purified source or 5% fetal bovine serum in the media. BB94, a synthetic metalloproteinase inhibitor, and HGF were included in the media as indicated. After the intended period of incubation (4–48 h), media were collected and cleared of cell debris by centrifugation and analyzed by SDS-PAGE impregnated with gelatin (1 mg/ml) as described (12) . For immunoprecipitation and Western blot, cells in 6-well plates were lysed in 250 µl of RIPA buffer (50 mM Tris, pH 7.5, 150 mM NaCl, 0.25% sodium deoxycholate, 0.1% Nonidet P-40, 10 µM leupeptin, 0.1 µM 5-APMSF, 1 µM aprotinin) supplemented with 10 mM EDTA to protect the active forms of MT3-MMP from degradation. The lysates were centrifuged at 14,000 g for 15 min to remove cell debris. Rabbit polyclonal anti-MT3-MMP antiserum (2 µl) was added to the resulting supernatants and incubated at 4°C for 1 h. The immune-complex was collected with protein-A/G PLUS agarose (10 µl, Santa Cruz), washed with RIPA buffer four times, then eluted with 2X SDS-PAGE sample buffer under reducing conditions. After electrophoresis, the proteins were transferred to PVDF membranes and probed with M2 anti-FLAG mouse monoclonal antibody and developed as described (28) .

Northern blot
Total RNAs were isolated from cells with TRI-REAGENT as suggested by the manufacturer (MRC, Cincinnati, Ohio). Equal amounts of total RNAs (10 µg) were denatured with glyoxal and DMSO, fractionated on a 1% agarose gel in 10 mM phosphate buffer at a constant voltage of 55 V for 5 h, and then transferred to nylon membrane overnight. The membrane was then stained with methylene blue for the 28s and 18s rRNA to establish equivalence in sample loading. The membrane was prehybridized at room temperature for at least 30 min, hybridized at 62°C for 16–24 h with (32) [32P]-labeled MT3-MMP cDNA as a probe, washed and exposed to an ABI screen, and scanned on a PhosphorImager (ABI, Foster City, Calif.).

Cell surface labeling with biotin
Cells were grown in 6-well plates and washed with ice-cold PBS three times before Sulfo-NHS-Biotin (Pierce, Rockford, Ill.) was added at 0.5 ml/well (0.5 mg/ml) and allowed to incubate for 1 h on ice. After extensive washing with a buffer containing 10 mM Tris, pH 7.4, 0.4 M Sucrose, 10 mM Glycine, 1.5 mM CaCl2, 5 mM MgCl2 for 10 min and ice-cold PBS, cells were lysed and immunoprecipitated as described above with anti-MT3-MMP antibody. The immunoprecipitates were divided into two fractions and analyzed by Western blots with either M2 anti-flag antibody or streptavidin conjugated with alkaline phosphatase (Pierce), respectively.

Deglycosylation
MT3-MMP proteins were immunoprecipitated as described above and the immune-complexes were eluted in 1% SDS and 5% 2-mercaptoethanol by boiling at 100°C for 10 min. The eluted materials were treated with or without Glycanase F for 15–20 h at 37°C in 20 mM sodium phosphate pH 8.0, 30 mM EDTA, 0.5% Nonidet P-40, 0.1% SDS, and 0.5% ß-mercaptoethanol as suggested by the supplier (Roche, Indianapolis, Ind.). The fractions were subsequently analyzed by Western blot using M2 antibody as described in previous sections.

Scattering assay
The scattering assay for MDCK cells was performed according to Stoker et al. (31) . Briefly, MDCK and its derivatives were seeded (10,000 cells/ml) in a 24-well tissue culture plate (1 ml/well). Cells were allowed to attach for at least 4 h, and HGF was added at indicated concentrations. Pictures were taken after 16 h of incubation as described below.

Growth and tubulogenesis of MDCK cells in 3-D collagen gel
Cells (1.2x103) were mixed with 250 µl of collagen (2 mg/ml; Collaborative, Waltham, Mass.) and allowed to gel at 37°C in 24-well plates giving rise to a three-dimensional (3-D) collagen matrices. Fresh media containing 95% DMEM and 5% fetal bovine serum were added to the wells with or without HGF (20 ng/ml) and changed every 2 days. After 12 days (MDCK, MT3-MMP wild-type, and EA mutant transfectants) or 6 days (MDCK and MT1-MMP transfectants), the growth and tubulogenesis of MDCK and its derivatives were photographed by a video camera attached to a Nikon microscope at the University of Minnesota Bioimaging Processing facility. Growth of MDCK cells in 3-D collagen gel is estimated by the diameter of the cysts. Depletion of gelatinases from FBS was accomplished by passing 10 ml of serum through a 5-ml gelatin-sepharose column (Sigma). The depleted serum was monitored by gelatin zymography as described above and sterilized by filtration (0.2 µm filter; Millipore, Bedford, Mass.). Gelatinase-depleted FBS was used for both growth and tubulogenesis assay as described above.


   RESULTS
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
DISCUSSION
 REFERENCES
 
The experimental model: a relationship between MMP activity and cellular growth/tubulogenesis in type I collagen gels
Based on the Kadono study, proteolytic activity, most likely contributed by MT1-MMP, is required for HGF-induced tubulogenesis of MDCK cells in 3-D collagen gels (27) . Given its well-defined multicellular organ-like structure (Fig. 1B ), this model system may be exploited to dissect the mechanism by which proteolytic enzymes are used by normal cells in basic development or tumor cells for invasion and metastasis. As initially reported, MMP inhibitor BB94 severely retarded the tubule-forming ability of HGF-induced MDCK cells in 3-D collagen gels (Fig. 1C ) (27) . Although the effect of BB94 could be interpreted as a requirement for MMP-mediated proteolysis against type I collagen matrix, it is equally possible that metalloproteinase activity is required for HGF signaling as reported for EGF receptor signaling (32) . We tested this possibility in an HGF-induced scattering assay on two-dimensional (2-D) surface, a process without the involvement of collagen degradation. Indeed, as shown in Fig. 1D , E , F , BB94 did not affect HGF-induced scattering of MDCK cells on 2-D collagen surface, suggesting that BB94 does not interfere with HGF signaling. Consistently, detailed observations of the BB94-inhibited and HGF-induced MDCK cells reveal that they are able to branch out (indicative of HGF signaling) but unable to expand into tubules (Fig. 1C ). Thus, MMP-mediated proteolysis is required for tubule formation, but not scattering.



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  		Figure 1. BB94 inhibits the tubulogenesis, but not the scattering response, of MDCK cells induced with HGF. In tubulogenesis assay, MDCK cells (1000/well) were embedded in 3-D collagen gels and cultured in 95% DMEM and 5% FBS either alone (A) or with 20 ng/ml of HGF (B, C) in the absence or presence of 10 µM of BB94 as indicated. Media were changed every 2 days. After 12 days, the cells were photographed as described in Materials and Methods. As for scattering, MDCK cells were seeded (10,000 cells/ml) in a 24-well tissue culture plate in 95% DMEM and 5% FBS medium (1 ml/well) (D–F). Cells were allowed to attach for at least 4 h, and 50 ng/ml of HGF with DMSO (E) or with 10 µM of BB94 (F) were added. Pictures were taken after 16 h of incubation. The experiments were repeated four times and the representative pictures were shown.

Generation and characterization of stable MT3-MMP transfectants
Although MT1-MMP may be the primary endogenous enzyme responsible for HGF-induced MDCK tubulogenesis in vitro, it is not clear whether other members of the MT-MMP subfamily also play an important role in kidney physiology and pathology in vivo. Based on heightened expression of MT3-MMP by invasive tumors, it has been hypothesized that MT3-MMP mediates ECM degradation and confers an invasive phenotype to tumor cells (7 , 20 , 21 , 23 , 25 , 33 , 34) . Interestingly, Kitagawa and colleagues have identified MT3-MMP among the MT-MMPs examined as a potentially important factor in the development of kidney carcinogenesis (23) . Taking these evidences together, we rationalize that the MDCK tubulogenesis model system is ideal and relevant for the analysis of MT3-MMP and its contribution of cellular invasiveness.

To analyze the role of MT3-MMP in this model system, we first isolated MT3-MMP and cloned its coding frame into the expression vector pCR3.1 with a FLAG tag at its carboxyl terminus (Fig. 2A ) (7 , 18 , 25) . Stable transfectants were generated in MDCK cells and characterized by Western blotting (12 , 28) . Among more than 20 MT3-MMP positive clones obtained, three representative clones with low (FF4–10), medium (FF4–2), and high (FF4–7) levels of expression as demonstrated by Northern blot were chosen for further characterization (Fig. 2B ). Consistently, these three clones express different levels of MT3-MMP protein on immunoblots with M2 antibody (Fig. 2C ). To clarify the nature of species in Figure 2C , the cell lysates were first immunoprecipitated with rabbit anti-MT3-MMP antibody, then blotted with M2 antibody against FLAG tag. As shown in Figure 2D , four MT3-MMP protein species were detected in all three clones at levels consistent with that of the Northern blot analysis in Figure 2B . Potentially, these multiple bands could be generated by differential glycosylation or/and zymogen activation. To differentiate these two possibilities, the samples from FF4–2 were treated with Glycanase F to remove N-glycosylations, which results in the reduction of four bands at 73, 70, 67, and 65 kDa (Fig. 2E , lane 1) to two at 68 and 65 kDa (Fig. 2E , lane 2). However, the signals for MT3-MMP bands were relatively weak, perhaps a result of autolysis (25) . Thus, BB94 was added to the culture media for the protection of MT3-MMP polypeptides from autolysis. Indeed, all four bands were enhanced by BB94, especially the 67/65 doublets (Fig. 2C , E , lanes 1, 3 and 2, 4, respectively). Given the fact that MT3-MMP possesses a furin-recognition signal for processing, it is reasonable to assign the deglycosylated 68-kDa and 65-kDa species as pro- and active species, respectively.



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  		Figure 2. Characterization of MT3-MMP stable transfectants. A) A schematic illustration for expression vector pCR3.1MT3-MMP. Shown are various domains for MT3-MMP (7) with a carboxyle-terminal FLAG tag (F). Pro, prodomain; Cat, catalytic domain; H, hinge; Pex, hemopexin-like domain; S, stem; T, transmembrane; C, cytosolic domain; F, FLAG. B) Northern blot analysis of MT3-MMP transfectants. Total cellular RNAs (10 µg/lane) from MDCK (lane 1), MDCK transfected with vector (lane 2), and three colonies of MT3-MMP transfectants (lanes 3–5) were fractionated, transferred, and hybridized as described in Materials and Methods. The 28S and 18S rRNAs were stained to show equivalent loading per lane (lower panel). The size for recombinant MT3-MMP transcript is ~2.5 kb. C) Detection of MT3-MMP by western blotting. Cell lysates from MDCK (lanes 1, 6), MDCK transfected vector (lanes 2, 7), and three colonies of MT3-MMP (lanes 3–5, 8–10) grown in 6-well plate with DMSO (lanes 1–5) or 10 µM BB94 (lanes 6–10) for 24 h were separated by SDS-PAGE, transferred, and blotted with anti-FLAG M2 antibody. The MT3-MMP–specific species are marked by brackets on the right. D) Identification of MT3-MMP by immunoprecipitation-western blotting. Cell lysates from MDCK (lane 1), MDCK transfected vector (lane 2), and three colonies of MT3-MMP (lanes 3–5) grown in 6-well plate with 10 µM BB94 for 24 h were immunoprecipitated with anti-MT3-MMP antisera, then were separated by SDS-PAGE, transferred, and blotted with anti-FLAG M2 antibody. The MT3-MMP–specific species are marked by brackets on the right. E) Deglycosylation of MT3-MMP proteins. FF4–2 cells were grown in 10-cm dishes to 50% confluence, treated with 5% FBS media containing either DMSO (lanes 1, 2) or 10 µM BB94 (lanes 3, 4) for 24 h. Cell lysates were immunoprecipiated with rabbit anti-MT3-MMP antisera and subjected to deglycolation, and analyzed by Western blot with anti-FLAG M2 antibody as described in Materials and Methods. Six bands of MT3-MMP proteins were marked as A, B, C, D, E, F and their molecular weights were shown in lane 5. Each band in panels 3 and 4 was quantified by a Photo documentation system from Stratagene (San Diego, Calif.) as 4383, 6618, 3170, 4713, 10754, 7872 units, respectively. Based on the intensities: A + B = ~E; C + D = ~F.

Processing of progelatinase A as a convenient indicator of MT3-MMP activity
Although the 65-kDa species is consistent with that of active MT3-MMP, it is not clear whether the transfected MT3-MMP exhibits any activity. One of the known functions for the MT-MMPs is their ability to process progelatinase A by cleaving the Asn (37) -Leu bond within the prodomain (15 , 18) . Therefore, processing of progelatinase A could serve as an accurate indicator of MT-MMP activities. As shown in Figure 3 , MT3-MMP transfectants FF4–2 and FF4–7 process ~90% of the progelatinase A in the media containing 5% FBS into the intermediate form in a BB94-sensitive fashion (lanes 3–6), whereas control transfected MDCK cells do not (lanes 1 and 2). These data strongly suggest that the transfected MT3-MMP is displayed on cell surface as active species, most likely the 65-kDa species in Figure 2 . However, the final activation step from the intermediate form to the active one was not observed, probably because of the presence of {alpha}2-macroglobulin in the fetal bovine serum, which can trap any active proteinase including active gelatinase A (35) . However, similar results were obtained when purified progelatinase A was incubated with the cells, thus, suggesting that the activation process is not influenced by serum components in the media (data not shown). We then attempted intracellular activation of progelatinase A by transfecting the cells with a gelatinase A expression vector. Contrary to cell-surface–mediated processing, a full range of activation products were observed for the transfected gelatinase A (Fig. 3 , lanes 9 and 11). Interestingly, BB94, which completely blocks the activation of progelatinase A from serum (extracellular), is only partially effective against co-expressed MT3-MMP and progelatinase A (Fig. 3 , lanes 10 and 12), suggesting that the intracellular pool of MT3-MMP is somehow not accessible to BB94 inhibition as reported (36) . Because control transfected MDCK cells cannot activate or process progelatinase A (Fig. 3 , lanes 1 and 7), it is concluded that transfected MT3-MMP is solely responsible for the progelatinase A processing activity observed in FF4–2 and FF4–7 cells.



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  		Figure 3. Activation of progelatinase A by MT3-MMP transfectants. MDCK transfected with control vector (lanes 1, 2, 7, 8) and two colonies of MT3-MMP transfectants named as FF4–2 (lanes 3, 4, 9, 10) and FF4–7 (lanes 5, 6, 11, 12) were allowed to activate the progelatinase A from the 5% FBS in DMEM medium (lanes 1–6) or progelatinase A by transfection with a pCR3.1MMP-2 vector (lanes 7–12), in the presence of DMSO (lanes 1, 3, 5, 7, 9, 11) or 10 µM BB94 (lanes 2, 4, 6, 8, 10, 12) for 48 h. Aliquots (5 µl) of the conditioned medium (500 µl for a well in the 6-well plate) were analyzed by gelatin zymography (8.5%, incubation for 12 h at 37°C). GelB, progelatinase B; GelA, progelatinase A. Note the pro (top), intermediate (middle), and active (bottom) bands for gelatinase A in lower panel and the pro (top) and intermediate (bottom) bands for gelatinase A in upper panel.

MT3-MMP enhances growth and tubulogenesis of MDCK cells in 3-D collagen lattice
To test the hypothesis whether MT3-MMP enhances tubulogenesis, a process involving invasion into the neighboring ECM, cells transfected with control or MT3-MMP expression vectors were inoculated into type I collagen gel and allowed to grow with or without HGF induction and in the presence or absence of MMP inhibitor BB94. Surprisingly, MT3-MMP transfectants grow into larger cysts without HGF and bigger tubules with HGF than control transfected MDCK cells in a BB94-sensitive fashion (Fig. 4A , panels A–H), suggesting that MT3-MMP–mediated proteolysis contributed to the growth and tubulogenesis of MDCK cells cultured in type I collagen gels. The diameters of the cysts were measured and presented as growth indices for MDCK cells as shown in Figure 4B . Apparently, MT3-MMP allowed the cells to grow into cysts ~50% larger than the control transfected MDCK cells (Fig. 4A , panels A and E; Fig. 4B ). Consistently, BB94 inhibited the growth of both control and the MT3-MMP transfectants by ~40% (Fig. 4A , panels A, B, E, F; Fig. 4B ). A similar trend of reduction in cell numbers is clearly discernible in HGF-induced culture in the presence of BB94 (Fig. 1 , panel C; Fig. 4A , panels B, D, F, H). Because MT1-MMP has been implicated as the endogenous enzyme responsible for tubulogenesis, we also tested whether similar growth enhancement can be observed (27) . Indeed, overexpression of MT1-MMP increased the size of cysts in type I collagen gel, which is also inhibited by BB94 (Fig. 4A , panels I and J) (27) . However, MT1-MMP transfectants are not able to form bigger or better tubules when induced by HGF, perhaps because of excessive degradation of type I collagen (Fig. 4A , panels K and L) (27) . Interestingly, when MT5-MMP was analyzed, the results were similar to those of MT3-MMP (X. W. and D. P., unpublished results).



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  		Figure 4. MT3-MMP enhances the growth and tubulogenesis of MDCK cells induced by HGF. A) MDCK cells (A–D, M–P), FF4–2 cells (E–H, QT), and MT1-MMP transfectants (MT1) (I–L) (1,000/well) were embedded in type I collagen (24-well plates) and allowed to grow in 5% FBS DMEM medium (A–L) or 5% gelatinases depleted FBS DMEM medium (M–T) with DMSO (A, C, E, G, I, K, M, O, Q, S) or 10 µM of BB94 (B, D, F, H, J, L, N, P, R, T), with (C, D, G, H, K, L, O, P, S, T) or without (A, B, E, F, I, J, M, N, Q, R) HGF induction (20 ng/ml). Media (500 µl/well) were changed every 2 days. After 6 or 12 days, cells were photographed as described in Materials and Methods. The experiments were repeated four times with the representative pictures shown here. B) Ten cysts in each group in panels A, B, E, F, of 4A were measured from the photographs and the mean values (4.36±0.43x10–2 cm as 100±9.9 for panel A; 2.58±0.34x10–2 cm as 59.2±7.8 for panel B; 6.59±1.03x10–2 cm as 151.1±23.6 for panel E; and 3.48±0.42x10–2 cm as 79.8±9.6 for panel F) were graphed. The vertical bars indicate standard errors. C) Depletion of gelatinases from FBS. Ten milliliters of FBS (lane 1) was loaded into a 5-ml gelatin-sepharose column and collected as the flow-through fraction (lane 2). Five microliters of each sample was analyzed by gelatin zymography (7.5%, incubation for 12 h at 37°C). GelB, progelatinase B; GelA, progelatinase A.

Because the experiments were carried out in the presence of serum, a rich source of progelatinase A, the effects of MT3-MMP might be indirect (i.e., by activating progelatinase A). To test this possibility, we repeated the same experiments with FBS depleted of gelatinase A by gelatin-sepharose column (Fig. 4C ). The resulting serum supported the growth and tubulogenesis of MDCK, FF4–2, as well as other MT3-MMP transfectants, almost indistinguishable from the original serum (Fig. 4A , panels M–T). Because MDCK cells do not express any endogenous progelatinase A (data not shown), it appears that MT3-MMP enhanced the growth and tubulogenesis of MDCK cells by mediating proteolysis directly, rather than by its ability to activate progelatinase A as proposed for MT1-MMP (4) .

HGF stimulates the activity of transfected MT3-MMP
To further explore the mechanism by which MT3-MMP enhances HGF-induced tubulogenesis, we investigated whether HGF influences the activity of the transfected MT3-MMP. Because progelatinase A activation is a reliable indicator of MT3-MMP activity (Fig. 3) , we measured MT3-MMP activity by assessing the activation of progelatinase A present in culture media containing 5% fetal bovine serum. As shown in Fig. 5A , HGF further stimulated FF4–2’s ability to process progelatinase A in a time-dependent manner (lanes 3 vs. 1, 7 vs. 5, 11 vs. 9, 15 vs. 13). At each time point, HGF-treated culture exhibits a higher ratio between active and latent gelatinase A than control FF4–2 cells, in an accelerating trend toward the 48th h when the ratio for HGF-treated culture is almost twice that of the control (Fig. 5B ). It is of interest to note that a small portion of the progelatinase B is also activated, especially at the 48-h time point (Fig. 5A ). Given that HGF-stimulated MDCK cells can also mediate minor activation of progelatinase B, it is very unlikely that MT3-MMP is responsible for the observed activation of progelatinase B (Fig. 5A , lanes 17 and 19). As expected, BB94 inhibited the activation process for both progelatinase A and B (Fig. 5A , lanes 2, 4, 6, 8, 10, 12, 14, 16, 18, 20). Thus, HGF seems to be able to enhance the activity of the transfected MT3-MMP, albeit quite unexpectedly. Mechanistically, this enhancement could be the result of 1) enhanced expression of the transfected gene, 2) alteration of TIMP levels, and 3) accelerated activation of MT3-MMP. We ruled out the first option by performing Northern blot analysis and found that MT3-MMP mRNA stayed the same with or without HGF (Fig. 5C ). Among TIMPs discovered so far, TIMP-2, but not TIMP-1, has been shown to stimulate MT1-MMP–mediated activation of proMM-2 at low concentrations, but inhibit the same process at higher concentrations (15) . However, MDCK cells appear to express no detectable TIMP-2 with or without HGF treatment (data not shown), ruling out any potential role of TIMP-2 in the observed enhancement of proMMP-2 activation. Thus, it is likely that HGF enhances MT3-MMP activity by promoting its zymogen activation. Indeed, as shown in Figure 5D , HGF seems to have accelerated the conversion of proMT3-MMP into the active forms as more 67/65-kDa species were detected in HGF-induced FF4–2 cells, especially when the active species were protected from autolysis by BB94 (25) (lanes 2–5). Furthermore, when cell-surface MT3-MMP was examined by biotin-labeling, more active MT3-MMP was detected on cell surface on HGF stimulation (Fig. 5D , lanes 8 and 10 vs. 7 and 9). These evidences support the conclusion that MT3-MMP is converted into active form and displayed on cell surface. More importantly, HGF seems to be able to accelerate the activation process of MT3-MMP and facilitate its surface presentation.



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  		Figure 5. The effects of HGF on MT3-MMP activity and progelatinase A activation. A) Time-dependent enhancement of progelatinase A activation by HGF. MDCK and FF4–2 (75% confluence) were incubated with 500 µl/well of 5% FBS DMEM medium with DMSO, 50 ng/ml of HGF, 10 µM BB94 or 50 ng/ml of HGF and 10 µM BB94 for 8 (lanes 1–4), 12 (lanes 5–8), 24 (lanes 9–12), 48 (lanes 13–20) h as indicated at both top and bottom. The supernatants were analyzed by zymography as described above. Shown here are representatives of three independent experiments. B) Ratio of active/pro forms of gelatinase A is enhanced in a time-dependent fashion by HGF. The amounts of pro and active gelatinase A from experiments in panel A were quantified by the intensities of the reverse imagines. The top band is considered the pro form and the bottom two as its processed forms. The mean values (n=3) for 8 (lanes 1, 3), 12 (lanes 5, 7), 24 (lanes 9, 11), 48 (lanes 13, 15) h were 0.28 ± 0.03, 0.57 ± 0.04, 1.11 ± 0.12, and 1.59 ± 0.15 for FF4–2 alone or 0.40 ± 0.03, 0.74 ± 0.03, 1.77 ± 0.04, and 2.90 ± 0.42 for FF4–2 induced by HGF, respectively. C) HGF does not increase mRNA expression of transfected MT3-MMP. FF4–2 cells were grown in 6-well plates in 5% FBS DMEM medium to ~75% confluence then were changed to 5% FBS DMEM (500 µl) with 50 ng/ml of HGF for incubation 0, 8, 12, and 24 h in 37°C as indicated. At each time point, total RNAs were isolated and analyzed by Northern blot as described above. Each lane was loaded with 10 µg of total RNAs. D) HGF increases the active form of MT3-MMP on cell surface. MDCK (lanes 1, 6) and FF4–2 (lanes 2–5, 7–10) cells were grown in 6-well plates to ~75% confluence, then treated with 5% FBS DMEM in the presence of DMSO (lanes 2, 7), 10 µm of BB94 (lanes 3, 8), 50 ng/ml of HGF (lanes 4, 9), or 50 ng/ml of HGF and 10 µm of BB94 (lanes 5, 10). After 24 h, the cell surfaces were labeled with biotin and lysed. The immunoprecipitates with anti-MT3-MMP antisera were either probed with M2 anti-flag antibody (lanes 1–5) or streptavidin conjugated with alkaline phosphatase (lanes 6–10) as described in Materials and Methods.

Catalytic activity of MT3-MMP is required for progelatinase A activation and its enhancement of MDCK growth and tubulogenesis
Although a correlation has been established between the expression of MT3-MMP active species and the enhancement of growth and tubulogenesis, it is not clear whether the proteolytic activity of MT3-MMP is required. To directly prove a causal relationship, we constructed a catalytically inactive mutant, MT3-MMPEA, by mutating the Glu residue in the HE247LGH catalytic motif (Fig. 6A , see Materials and Methods). Stable transfectants were generated for MT3-MMPEA and assayed for its ability to activate progelatinase A. As shown in Figure 6B , cells generated from MT3-MMPEA failed to activate progelatinase A, while expressing the mutant proteins at a relatively high level (Fig. 6C ). The relatively higher level of MT3-MMPEA is expected because BB-94 can protect wild-type MT3-MMP from autolysis (Fig. 2) . Consequently, in both growth and tubulogenesis assays, the MT3-MMPEA mutants behave similarly as the control transfected MDCK cells, with slower growth and smaller tubules when compared with FF4–2 (Fig. 6D ). Together, these data demonstrate strongly that the proteolytic activity of MT3-MMP is required for the observed enhancement of both growth and tubulogenesis of MDCK cells.



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  		Figure 6. Catalysis is required for MT3-MMP–enhanced growth and tubulogenesis of MDCK cells. A) Schematic diagrams of wild-type and catalytically inactive mutant of MT3-MMP. MT3-MMPEA is the same as the wild type except a single change of Glu247 to Ala. B) MT3-MMPEA fails to activate progelatinase A. MDCK transfected vector (lanes 1, 2), FF4–2 cells (lanes 3, 4), and MT3-MMPEA cells (lanes 5, 6) were assayed for the activation of progelatinase A from the culture media in the absence (lanes 1, 3, 5) or presence (lanes 2, 4, 6) of 10 µM BB94 for 24 h as described above. Gel A, gelatinase A; GelB, gelatinase B; MT3EA, MT3-MMPEA. C) MT3-MMPEA is synthesized and activated as the wild-type molecule. Control transfected MDCK (lane 1), FF4–2 cells (lane 2), and MT3EA cells (lane 3) were grown in 6-well plate with 10 µM BB94 for 100% confluence, then analyzed by immunoprecipitation and western blotting as described in Fig. 2D . Note the enhanced stability of MT3-MMPEA versus wild type. D) MDCK cells (A, B), MT3-MMPEA stable transfectant (C, D), and FF4–2 cells (E, F) (1,000/well) were embedded in type I collagen and assayed for growth and tubulogenesis as described above. After 12 days, the cultures were photographed. Shown are representative of two experiments.


   DISCUSSION
TOP
 ABSTRACT
 INTRODUCTION
 MATERIALS AND METHODS
 RESULTS
 DISCUSSION
REFERENCES
 
Among the MMPs discovered so far, the membrane-type matrix metalloproteinases (MT-MMPs) have been proposed as versatile enzymes critical for a wide range of biological processes, underscored by the recent finding that MT1-MMP is the first to exhibit a dramatic phenotype among MMP knock-out (KO) mice (17) . Despite intense interest in these enzymes, little is known about the mechanisms by which MT-MMPs exert their proteolytic activity to specify a particular phenotype. While searching for a relevant experimental system for analyzing MT3-MMP, an enzyme known to be expressed by malignant tumors including renal carcinomas, we have adopted a well-defined in vitro model: HGF-induced tubulogenesis of renal cell line MDCK in type I collagen gels, a process previously shown to be MT1-MMP dependent (23 , 27) . We demonstrated that the transfected MT3-MMP is expressed and converted into active species, displayed on cell surface, and able to promote the growth and tubulogenesis of MDCK cells in type I collagen matrix in a manner independent of its hallmark function—activation of progelatinase A. To our knowledge, this is the first study attempting to establish a relationship between the basic biochemical properties of MT3-MMP and any cellular phenotype in a physiologically relevant environment: 3-D type I collagen lattice as opposed to plastic surface. By demonstrating MT3-MMP’s ability to modify the cell-matrix microenvironment of the recipient cells and allow them to grow and form organized structures, we hope that this model could be adopted as one of the standard model systems for analyzing MMP functions, especially for those of MT-MMPs. With relative ease of generating stable lines and scoring phenotypes such as growth and tubulogenesis, one can subject all MT-MMPs under the same experimental conditions to compare and contrast their biochemical properties and contributions to growth and tubulogenesis as described in this study. The detailed mechanistic insights generated from this experimental approach may help establish the functional basis of MT-MMP–related phenotypes in complex systems such as knock-out mouse models or human malignant diseases. Given the fact that MT3-MMP is up-regulated significantly in human renal carcinoma tissues (23) , our findings provide strong support to the idea that MT3-MMP is expressed by tumor cells to gain growth and invasive advantages over their surrounding tissues, which are filled with densely packed extracellular matrix such as type I collagen. As discussed below, a continued effort to dissect the mechanistic contribution of MT3-MMP in this model system may generate a useful working model for MT-MMP activities, and their contributions to cellular growth and morphogenesis within physiologically relevant matrix such as type I collagen gels.

MT3-MMP confers a phenotypic response to MDCK cells
MT3-MMP has been characterized as an enzyme capable of activating progelatinase A and degrading many ECM components including type III collagen (7 , 24 , 25) . Consistent with its role in ECM remodeling processes, MT3-MMP has been detected in a number of physiological as well as pathological conditions such as tumor progression (23 , 37) , microglial cells (20) , and smooth muscle cells (25) . In this study, we demonstrated that MT3-MMP enhances the ability of MDCK cells to grow and form tubules in 3-D type I collagen gels when induced by HGF. Although MT3-MMP can not degrade type I collagen into typical one- and three-quarter fragments, it can cleave the Gly4-Ile5 bond within the triple helical portion of alpha2 (I) chains (24) . This activity may be more critical in supporting the tubulogenesis phenotype of MDCK cells than the one- and three-quarter degradation activity. Indeed, when expressed in MDCK cells, MT1-MMP, known for its activity to degrade type I collagen into one- and three-quarter forms, can enhance the growth of MDCK cells as cysts, but actually inhibits the tubulogenesis process, because of its excessive degradation of type I collagen (Fig. 4A ) (26) . Initially, we were surprised to observe that MT1-MMP transfectants did not enhance tubulogenesis as reported (26) . Considering the delicate balance between matrix destruction and tubular formation, it is highly sensible that only the right amount of proteolytic activity can enhance tubulogenesis, reflecting perhaps a more realistic and balanced scenario in vivo.

Collagen as a barrier for growth
Type I collagen has been used extensively as a barrier for assaying tumor invasion in vitro (38 39 40) . Our data demonstrated that type I collagen could also inhibit the growth of embedded cells. First, MMP inhibitor BB94 blocked the growth of MDCK cells as cysts in collagen lattice (Fig. 4) . Secondly, more MMP activity, provided in the form of MT3-MMP and MT1-MMP expression, enhanced the growth of the transfectants significantly (Fig. 4) . Given the fact that BB94 has no effect on the growth of cells on top of type I collagen or plastic surfaces, it is very unlikely that the inhibitor affects cell proliferation or collagen-mediated signaling (Fig. 1) (41) . In agreement with our findings, collagen lattice has been shown to inhibit the growth of dermal fibroblasts (38) . Perhaps, this finding is relevant to tumorigenesis because the initial transformation of tumor cells requires more space for them to grow and expand. Indeed, recent attention has been shifting to the roles of MMPs in interstitial malignancies by clearing more growing space (42) . We suggest that the roles of MMPs in tumorigenesis should be broadened to earlier phases of carcinogenesis in addition to the current emphasis on tumor invasion and metastasis.

Multiple forms of MT3-MMP and surface localization of its active forms
As demonstrated with the catalytically inactive MT3-MMPEA, intrinsic proteolytic activity of MT3-MMP is required for its ability to enhance MDCK growth and tubulogenesis in collagen lattice (Fig. 6D ). Like other MMPs, MT3-MMP must undergo zymogen activation before expressing any proteolytic activity. Indeed, both pro and activated forms of MT3-MMP were detected in total cell lysates (Figs. 2D , E and 5D ). Interestingly, only the active form of MT3-MMP was detected on cell surface (Fig. 5D ). By monitoring progelatinase A activation, we can measure quite accurately the activity of MT3-MMP in cell culture (Figs. 3 , 5A ). Thus, this experimental system reconstitutes all known functions of MT3-MMP. We were surprised by the fact that only active forms of MT3-MMP (67/65 kDa) were identified on cell surface by biotin labeling (Fig. 5D ) without any significant amount of prospecies. This is apparently in agreement with the fact that the MT1-MMP initially purified from HT1080 cells and recombinant vaccinia virus-based system is in active form with processed NH2 terminus at Tyr-Ala-Ile- (15 , 43) . The failure to detect the proenzymes at the cell surface suggest that processing by furin or proprotein convertases is perhaps a prerequisite for the trafficking of MT-MMPs to the cell surface, thus providing an important mechanism of regulation for MT-MMPs. Thus, as transmembrane proteinases, it is expected that MT-MMPs be regulated at multiple stages from transcription to trafficking.

Progelatinase A activation and MT3-MMP–mediated cellular functions
The activation of progelatinase A by MT1, 2, 3, 5-MMPs has become a standard function for these membrane-bound enzymes and is believed to be the mechanism by which they exert any biological function (4 , 7 , 8 , 12) . Evidence presented here supports the notion that MT3-MMP alone can enhance the growth and tubulogenesis of MDCK cells, requiring no participation of progelatinase A (Fig. 4A ). This conclusion is certainly consistent with the recent findings that MT-MMPs possess intrinsic proteolytic activities against a wide range of substrates, thus, are able to remodel the ECM independent of progelatinase A (18 , 25) . With the expansion of the MT-MMP family, it is highly unlikely that the sole function of at least four MT-MMPs is to generate active gelatinase A. Instead, we propose that MT-MMPs could remodel ECM directly or in concert with their downstream proteinases such as progelatinase A and procollagenase 3 (17 , 44 45 46 47) . Further studies are needed to delineate the relative contribution of these strategies for MT3-MMP or its related members in the destruction of ECM during development and diseases processes.


   ACKNOWLEDGMENTS
 
The authors wish to thank Dr. R. Fridman (Wayne State University) for providing TIMP1, 2 and progelatinase A and Helen Mills of British Biotech for providing BB94. This study was supported in part by CA76308 from the National Cancer Institute, American Heart Association Grant-in-Aid 9750197N, Elsa Pardee Foundation, University of Minnesota grant-in-aid, Minnesota Medical Foundation.

Received for publication April 24, 2000. Revision received May 25, 2000.
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 RESULTS
 DISCUSSION
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